Preferential subcortical collateral projections of pedunculopontine nucleus-targeting cortical pyramidal neurons revealed by brain-wide single fiber tracing

The pedunculopontine nucleus (PPN) is a heterogeneous midbrain structure involved in various brain functions, such as motor control, learning, reward, and sleep. Previous studies using conventional tracers have shown that the PPN receives extensive afferent inputs from various cortical areas. To examine how these cortical axons make collateral projections to other subcortical areas, we used a dual-viral injection strategy to sparsely label PPN-targeting cortical pyramidal neurons in CaMKIIα-Cre transgenic mice. Using a high-speed volumetric imaging with on-the-fly-scan and Readout (VISoR) technique, we visualized brain-wide axonal projections of individual PPN-targeting neurons from several cortical areas, including the prelimbic region (PL), anterior cingulate area (ACA) and secondary motor cortex (MOs). We found that each PPN-projecting neuron had a unique profile of collateralization, with some subcortical areas being preferential targets. In particular, PPN-projecting neurons from all three traced cortical areas exhibited common preferential collateralization to several nuclei, with most neurons targeting the striatum (STR), lateral hypothalamic area (LHA) and periaqueductal gray (PAG), and a substantial portion of neurons also targeting the zona incerta (ZI), median raphe nucleus (MRN) and substantia nigra pars reticulata (SNr). Meanwhile, very specific collateralization patterns were found for other nuclei, including the intermediate reticular nucleus (IRN), parvicellular reticular nucleus (PARN) and gigantocellular reticular nucleus (GRN), which receive collateral inputs almost exclusively from the MOs. These observations provide potential anatomical mechanisms for cortical neurons to coordinate the PPN with other subcortical areas in performing different physiological functions. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-022-00975-y.


Animals
Adult male and female mice (8-16 weeks) were used in the experiment. Tracing experiments were performed using the CaMKⅡα-Cre line. All mice were group-housed with food and water accessible ad libitum under a 12-hour light/dark cycle (6:00-18:00).
All procedures were performed according to the guidelines of the Animal Use and Care Committee of the University of Science and Technology of China.

Tissue processing
Mice were deeply anesthetized and sacrificed 3-4 weeks after surgery, followed by transcardial perfusion with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer.
Then, the slices were immersed in Hoechst solution (10 µg/ml) for 4 hours. Finally, the samples were rinsed in 0.1 M PBS three times before being mounted.

VISoR imaging and image processing
Brain slices were mounted on a quartz slide with polymerized HMS. Slices were immersed in a refractive-index-matching solution with a refractive index of 1.46 for 8 hours. Fluorescent images of the whole brain were acquired using the VISoR system at a resolution of 1 × 1 × 2.5 µm 3 [3]. Image data were reconstructed automatically using custom software as previously described [4]. Adjacent slices were stitched together by nonrigid transformation followed by elastic deformation of local domains. Images were registered using custom software according to the Allen Mouse Brain Common Coordinate Framework (Allen CCF) [5]. In retrograde tracing experiments, cell counting was trained with ilastik software to identify and count the neurons in each 25 µm brain slice. These counting results were matched to the Allen CCF by custom software to obtain the number of neurons within each brain area. For fiber tracing, axons and dendrites were annotated semiautomatically using custom software implementing the Virtual Finger technology at 1-μm resolution. For validating the tracing pathway, each neuron was traced by two trained tracers and checked by the other more experienced annotator by merging the two tracing pathways. In total, we imaged 3 animals and traced the full morphology of 15 neurons, 11 of which were validated and used for further analyses.

Quantification and statistics
To assess whole-brain inputs to the PPN, the input proportion for each identified upstream brain area was calculated from the number of retrogradely labeled neurons in this area divided by the total number of labeled neurons in the whole brain. The mean and standard error of this value were calculated based on the data from 3 injected animals and were plotted in Fig. S2. For the single neuron collateralization analysis in Fig. 1i, the number of axonal termini (target number) in one brain area was used to quantify the density of the projection from each traced cortical neuron. For overall collateralization analysis of afferents received by different subcortical targets from the cortical areas (Fig. 1j), the collateralization ratio was defined as the number of traced neurons from a given cortical area, each having at least 2 termini in a specified subcortical target area divided by the total number of neurons traced for this cortical area. The same definition was also used to quantify search results from the mouse brain projectome database [7]. From the database, the number of PPN-projecting neurons was obtained by searching for neurons with soma located in the MOs, ACA or PL, while having at least two termini in the PPN. The number of neurons that also project to a collateralization target identified in Fig 1i was